Nucleic acid extraction apparatus

ABSTRACT

This invention provides an apparatus for extracting nucleic acids from nucleic acid-containing samples. The nucleic acid extraction apparatus of the invention comprises (1) a group of extraction vessels each comprising a reactor tube in which a sample, a reagent solution, and a magnetic carrier are admixed and reacted, a drain cup for pooling an unwanted component solution, and a nucleic acid recovery tube all as secured to a support, (2) a distribution means for introducing a solution into each of the extraction vessels, (3) a stirring means for mixing the solution and magnetic carrier in the reactor tube, (4) a holding means for holding the magnetic carrier stationary within the vessel, (5) a discharging means for discharging the solution from the reactor tube while the magnetic carrier is held stationary, (6) a heating means for heating the solution and magnetic carrier in the reactor tube, and (7) a transfer means for serially transferring the vessels to the given positions.

FIELD OF THE INVENTION

The present invention relates to an apparatus for extracting nucleicacids from various nucleic acid-containing samples, particularlybiological samples. More particularly, the invention relates to anucleic acid extraction apparatus well suited for a technology forextracting nucleic acids using a nucleic acid-binding magnetic carrier.

DESCRIPTION OF RELATED ART

The extraction of nucleic acids from biological or other samples isbeing practiced of late in many fields. For example, genetic engineeringand construction of DNA probes in particular involve a procedure ofextracting DNA or RNA coding for an objective protein from cellsproducing the protein. Moreover, the clinical examination for detectinga virus DNA or RNA using a DNA probe includes a procedure of extractingthe target DNA or RNA from a biological sample.

The procedure of extracting nucleic acids is, thus, of great importancein various fields. The hitherto-known technology for extracting nucleicacids includes a method which comprises treating a sample with a causticreagent, extracting the nucleic acid with phenol or phenol-chloroform,and recovering DNA by ethanol precipitation and a method which comprisestreating a sample with a proteolytic enzyme such as proteinase K in thepresence of a surfactant and then extracting the nucleic acid (IdenshiKogaku [Genetic Engineering], Vol. 1, No. 1, p. 112-119, 1997), amongothers.

The vessel used conventionally in those extraction methods includes agroup of plastic microtubes each provided with a cover as described inUnexamined Japanese Patent Publication No. 18362/1985 (Examined JapanesePatent Publication No. 31945/1992). This vessel is generally used withsaid cover kept open during the extraction procedure.

A variety of nucleic acid extraction apparatuses have been proposedaccording to the above nucleic acid extraction technology.

For example, Unexamined Japanese Patent Publication No. 125972/1991describes a nucleic acid extraction apparatus designed to prevent viralinfection and improve the efficiency of extraction which comprises amultiarticulated industrial robot and peripheral units necessary for DNAextraction and purification.

Unexamined Japanese Patent Publication No. 131076/1992 discloses anextraction apparatus designed to improve the efficiency of extraction ofnucleic acids from a small amount of blood or other biological materialthrough a compact arrangement of means for transfer of the nucleic acidextraction vessel to a centrifuge.

Unexamined Japanese Patent Publication No. 187077/1992 discloses anautomatic pretreatment system for DNA sequencing which comprises afilter unit attached to an aspiration-discharge system, a thermostat anda means for carrying out the necessary mixing and concentration.Unexamined Japanese Patent Publication No. 47278/1997 discloses anextraction apparatus employing a filter system equipped with a vacuumpump in lieu of a centrifuge.

The nucleic acid extraction procedure generally comprises a series ofsteps, namely hemolysis, leukocyte concentration, cell membrane lysis,and nucleic acid purification. The operations performed in theabove-mentioned extraction methods in common are illustrated in theblock diagram of FIG. 1. Thus, the nucleic acid extraction proceduregenerally comprises an operation for charging the vessels with samplesand other solutions, a mixing operation necessary for carrying out thereaction, a solid-liquid separating operation, and an operation forremoval of unwanted components or recovery of the nucleic acid.

Meanwhile, the conventional nucleic acid extraction apparatus describedabove is such that the solid-liquid separation in the above series ofoperations is performed either by means of a centrifuge or byfiltration. Therefore, in order that a fully automatic extractionapparatus may be implemented, a centrifuge or a vacuum pump and theassociated hardware must be built into the apparatus, with the resultthat the whole apparatus is inevitably bulky, complicated inconstruction, and increased in fabrication cost.

As the result of recent advances in the nucleic acid amplificationtechnology, it has become possible to analyze and detect the desireddomain of a nucleic acid using a very small amount of sample. Therefore,the technology for extracting nucleic acids from small biologicalsamples with high reliability is more important than the technology forextracting nucleic acids on a large scale.

In clinical examination or diagnosis, a large number of samples must bedealt with at one time. Therefore, it is common practice to perform anucleic acid extraction using a multiplicity of vessels arranged forrespective samples within the limited space of an automated apparatus.

In this case, however, when the above-mentioned so-called open-systemmicrotubes are used as the vessels and the distribution or dischargingof the samples, extraction reagents, etc. is carried out, thecontamination problem which is most abhorred in clinical diagnosis isliable to take place because of carryovers from one microtube toanother.

SUMMARY OF THE INVENTION

The present invention has for its primary object to provide an automaticextraction apparatus by which the extraction of nucleic acid involving acomplicated series of operations can be automatically performed.

It is a further object of the invention to overcome the drawbacks of theconventional nucleic acid extraction apparatuses, namely the increasedequipment size, complexity and high cost due to the use of a centrifugeor a vacuum pump and the above-mentioned contamination problem andthereby provide a nucleic acid extraction apparatus free from suchdisadvantages and suited for use in clinical diagnosis.

It is a still further object of the invention to provide an extractionvessel suited for use in the nucleic acid extraction method utilizing anucleic acid-binding magnetic carrier as described in UnexaminedJapanese Patent Publication No. 19292/1997, for instance, and a nucleicacid extraction apparatus tailored to the structural features of saidextraction vessel.

The present invention, thus, is directed to the following nucleic acidextraction apparatuses 1-6.

1. A nucleic acid extraction apparatus comprising

(1) a group of extraction vessels each comprising a reactor tube inwhich a sample, a reagent solution and a magnetic carrier are admixedand reacted, a drain cup for pooling an unwanted component solution anda nucleic acid recovery tube, all as secured to a supporting plate,

(2) a distribution means for dispensing a sample or other solution intoeach of said extraction vessels,

(3) a stirring means for stirring the sample solution and magneticcarrier within the extraction vessel,

(4) a holding means for holding said magnetic carrier stationary in aposition within the extraction vessel,

(5) a discharging means for discharging the sample solution from theextraction vessel while said magnetic carrier is held stationary,

(6) a heating means for heating the sample solution containing themagnetic carrier in said extraction vessel, and

(7) a transfer means for transferring said extraction vessel to therequired positions where said distribution means, stirring means,holding means, discharging means, and heating means are respectivelydisposed.

2. A nucleic acid extraction apparatus comprising

(1) a vessel stand adapted to support a group of extraction vessels eachcomprising a reactor tube to be filled with a magnetic carrier, a sampleand a reagent solution, a drain cup and a nucleic acid recovery tube,all as secured to a supporting plate;

(2) a distribution means for dispensing a sample or other solution intothe reactor tube of said extraction vessel installed on said vesselstand;

(3) a stirring means for stirring the magnetic carrier, sample, andreagent solution within the reactor tube;

(4) a holding means for holding the magnetic carrier stationary withinthe reactor tube;

(5) a discharging means for discharging the solution in the reactor tubeinto the drain cup or nucleic acid recovery tube while the magneticcarrier is held stationary within the reactor tube;

(6) a heating means for heating the reactor tube containing at leastsaid magnetic carrier;

(7) a transfer means for transferring the vessel stand carrying theextraction vessels horizontally; and

(8) a control means for controlling said respective means.

3. A nucleic acid extraction apparatus as defined in 2 wherein thecontrol means has a function to control the transfer means in such amanner the vessel stand may be transferred to any of the positions wheresaid distribution means, stirring means, holding means, dischargingmeans, and heating means are respectively disposed.

4. A nucleic acid extraction apparatus as defined in 2 furthercomprising a means for transferring the extraction vessels andinstalling them in an array on the vessel stand.

5. A nucleic acid extraction apparatus as defined in 2 wherein theextraction vessel for installing in the vessel stand comprising

a reactor tube, a drain cup, and a nucleic acid recovery tube as securedto a supporting plate,

a channel cover adapted to provide a connecting path between the nucleicacid recovery tube and the reactor tube as mounted in a top opening ofsaid recovery tube,

a cover with a hole for connection of an exhaust nozzle as mountedair-tight in a top opening of said drain cup,

a rotary element comprising a piercing pipe for insertion and connectionof a distribution nozzle, a valve passageway adapted to provide aconnecting path between the reactor tube and the drain cup or therecovery tube, and a rotating grip as mounted air-tight andaxial-rotably in a top opening of said reactor tube, and

one end of said valve passageway being connected liquid-tight to adischarging pipe extending axially within the reactor tube and the otherend being connected air-tight to a passageway communicating with theinterior of the drain cup.

6. A nucleic acid extraction apparatus as defined in 5 wherein at leastthe nucleic acid recovery tube of the extraction vessel is detachablefrom the reactor tube and drain cup.

The present invention is further directed to the following nucleic acidextraction vessels 7 and 8.

7. A vessel for nucleic acid extraction which comprises

a reactor tube, a drain cup, and a nucleic acid recovery tube as securedto a supporting plate,

a channel cover adapted to provide a connecting path between the nucleicacid recovery tube and the reactor tube as mounted in a top opening ofsaid recovery tube,

a cover with a hole for connection of an exhaust nozzle as mountedair-tight in a top opening of said drain cup,

a rotary element comprising a piercing pipe for insertion and connectionof a distribution nozzle, a valve passageway adapted to provide aconnecting path between the reactor tube and the drain cup or therecovery tube, and a rotating grip as mounted air-tight andaxial-rotably in a top opening of said reactor tube, and

one end of said valve passageway being connected liquid-tight to adischarging pipe extending axially within the reactor tube and the otherend being connected air-tight to a passageway communicating with theinterior of the drain cup.

8. A nucleic acid extraction vessel as defined above wherein at leastsaid nucleic acid recovery tube is detachable from said reactor tube anddrain cup.

Furthermore, the present invention provides the following nucleic acidextraction methods 9-12.

9. A method for nucleic acid extraction using the nucleic acidextraction apparatus of the invention which comprises

(a) a step of extracting a nucleic acid from a sample and causing it tobe adsorbed on a magnetic carrier in a reactor tube of an extractionvessel;

(b) a step of separating the magnetic carrier carrying the nucleic acidadsorbed thereon from the solution;

(c) a step of washing the separated magnetic carrier;

(d) a step of eluting the nucleic acid from the magnetic carrier, and

(e) a step of recovering the nucleic acid.

10. A method for nucleic acid extraction as defined above wherein said(a) extraction step, (c) washing step, and (d) elution step are carriedout by introducing an extraction solution, a wash solution, and aneluent, respectively, into the reactor tube containing the magneticcarrier by using the distribution means and then stirring the mixture byusing the stirring means.

11. A method for nucleic acid extraction as defined above wherein said(b) magnetic carrier separation step and (e) nucleic acid recovery stepcomprise discharging the solution from the reactor tube into the draincup and nucleic acid recovery tube, respectively, by using thedischarging means while the magnetic carrier is retained stationarywithin the reactor tube by using the holding means.

12. A method for nucleic acid extraction as defined above wherein said(d) nucleic acid elution step comprises drying the magnetic carriercarrying the nucleic acid adsorbed thereon by using the heating means,introducing an eluent into the reactor tube containing said magneticcarrier by using the distribution means, stirring the magnetic carrierin the eluent by using the stirring means, heating the solution by usingthe heating means, and stirring it by using the stirring means again.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing operations common to a generalprocedure for nucleic acid extraction.

FIG. 2 is a sectional view showing a nucleic acid extraction vessel foruse in the invention, wherein the reference numeral 1 represents areactor tube, 2 a drain cup, 3 a recovery tube, 4 a supporting plate, 5a discharging pipe, 6 a rotary element (valve), 7 a cover, 8 a channelcover, 9 a retaining cover, 10 a piercing pipe, and 11 a valvepassageway.

FIGS. 3a and 3 b are schematic views showing the rotary element (valve)6 of the nucleic acid extraction vessel, wherein the reference numeral10 and 11 indicate the same piercing pipe and valve passageway as shownin FIG. 2 and the numeral 12 represents a grip (ribs).

FIG. 4 is a sectional view showing a nucleic acid extraction vessel,wherein the element 6 has been axially rotated through 180 degrees fromthe position shown in FIG. 2. Each reference numeral represents the sameas shown in FIG. 2.

FIG. 5 is a view showing a nucleic acid extraction apparatus embodyingthe principles of the invention, wherein FIG. 5a is a top view and FIG.5b is a side view. In the views, the reference numeral 13 represents avessel stand, 14 a distribution means, 15 a stirring means, 16 adischarging means, 17 a holding/heating means, 18 a reagent bottle orbottle group, 19 a motor, 20 a continuous belt, and 21 a rail.

FIG. 6 is a schematic view showing a typical distribution means 14 foruse in the invention, wherein the reference numerals 23 and 24 eachrepresents a motor and the reference numeral 25 represents a rail, 26 alinear motor base, 27 a rotary arm, 28 a rotary arm grip, 29 a polescrew, 30 a tube, 31 a movable piece, 32 a spring, 33 an L-shaped metal,34 a distribution nozzle, and 35 a pad.

FIG. 7 is a schematic view showing a typical stirring means 15 for usein the invention, wherein the reference numeral 36 represents a ringmagnet, 37 a motor, 38 and 39 each a fixing metal, and 40 a pole screw.

FIG. 8 is a schematic view showing a typical unit 17 comprising aholding means 17 a and a heating means 17 b for use in the invention,wherein the reference numeral 41 represents a permanent magnet, 42 aheating block, 43 a mounting metal, 44 a platform, 45 a motor, 46 and 47each a fixing metal, and 48 a pole screw.

FIG. 9 is a schematic view showing a typical discharging means 16 foruse in the invention, wherein the reference numeral 41 represents apermanent magnet, 49 a motor, 50 a pole screw, 51 a movable piece, 52 anL-shaped metal, 53 a pressure nozzle, 54 a pad, 55 a tube, 56 a jointmetal, 57 an exhaust nozzle, and 58 a pad.

FIG. 10 is a flow-chart showing the nucleic acid extraction procedureusing the nucleic acid extraction apparatus of the invention.

FIG. 11 is a schematic diagram illustrating a distribution step in thenucleic acid extraction procedure using the nucleic acid extractionapparatus of the invention, wherein the reference numeral 34 representsa dispensing nozzle and 35 a pad.

FIG. 12 is a schematic view showing a stirring step in the nucleic acidextraction procedure using the nucleic acid extraction apparatus of theinvention, wherein the reference numeral 36 represents a ring magnet.

FIG. 13 is a schematic diagram illustrating a B/F separation step in thenucleic acid extraction procedure using the nucleic acid extractionapparatus of the invention, wherein the reference numerals 41 and 61represent a permanent magnet and magnetic silica beads, respectively.

FIG. 14 is a schematic diagram illustrating a step of discharging anunwanted component solution in the nucleic acid extraction procedureusing the nucleic acid extraction apparatus of the invention, whereinthe reference numeral 41 represents a permanent magnet, 53 and 54represent a pressure nozzle and a pad, respectively, and 57 and 58 anexhaust nozzle and a pad, respectively.

FIG. 15 is a schematic diagram illustrating a drying step or an eluentheating step in the nucleic acid extraction procedure using the nucleicacid extraction apparatus of the invention, wherein the referencenumeral 42 represents a heating block.

FIG. 16 is a schematic diagram illustrating a nucleic acid recovery stepin the nucleic acid extraction procedure using the nucleic acidextraction apparatus of the invention, wherein the reference numeral 41represents a permanent magnet and 53 and 54 represent a pressure nozzleand a pad, respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a nucleic acid extraction apparatusincluding a group of nucleic acid extraction vessels each comprising areactor tube, a drain cup for pooling an unwanted component solution,and a nucleic acid recovery tube as a unit, which is adapted to preventvessel-to-vessel carryover in the extraction procedure and henceeliminate the risk for contamination. In this specification, the abovevessel is referred to as “nucleic acid extraction vessel” or sometimesbriefly as “extraction vessel”.

Of the above extraction vessel, the reactor tube is a component unit inwhich the reaction necessary for treatment of a sample or analysisthereof for an objective component is carried out, the drain cup is acomponent unit in which the unwanted component solution formed by thereaction for said treatment or analysis is pooled, and the recovery tubeis a component unit in which the objective nucleic acid is recovered.

Furthermore, in this extraction vessel, the reactor tube, drain cup andrecovery tube are fixed in tandem to a supporting plate.

The sample which can be dealt with includes but is not limited tobiological materials from human or animal, such as the blood, serum,buffy coat, urine, feces, seminal solution, saliva, hair, sputum, andtissue cells (inclusive of cultured cells), as well as various othercells and cell cultures. Among them, preferred are biological materialsfrom human.

This invention is now described in detail, reference being had to theaccompanying drawings. The reference numerals used in the followingdescription correspond to the numerals used in the drawings.

1. Extraction vessel

FIG. 2 is a sectional side elevation view showing a typical extractionvessel for use in the nucleic acid extraction apparatus of theinvention.

The extraction vessel essentially comprises a reactor tube 1 in which asample is mixed and reacted with reagents etc., a drain cup 2 forpooling an unwanted component solution and a tube 3 for recovering thenucleic acid extracted from the sample, and those component units arefixed tandem to a supporting plate 4. It should be understood that thisfixation may optionally be a disconnectable one.

Of this extraction vessel, said three component units, namely thereactor tube, drain cup, and recovery tube, may be molded integrally orin such a manner that at least one of the component units may bedisconnected from the other units. It is preferably arranged so that atleast the nucleic acid recovery tube 3 may be detached from the otherunits, and even from the supporting plate 4, for example aftercompletion of nucleic acid extraction.

The drain cup 2 has a cover 7, the recovery tube 3 has a channel cover8, and the reactor tube 1 has a rotary element 6 (a valve), on the topopenings thereof. The rotary element 6 is secured to the reactor tube 1through a retaining cover 9 in such a manner that it may be rotatedabout the axis of the reactor tube 1. As installed within the reactortube 1, a discharging pipe 5 extending in the axial direction of thereactor tube is connected liquid-tight to a valve passageway 11 in saidrotary element 6. Preferably, the lower end of the discharging pipe 5and the inner bottom of the reactor tube 1 are spaced apart by about1-10 mm.

FIG. 3 shows the rotary element 6. FIG. 3a is a top view of the rotaryelement and FIG. 3b is a side elevation view thereof. The rotary element6 is connected air-tight to the top of the reactor tube 1 and providesvarious passageways. The rotary element 6 comprises a piercing pipe 10,a valve passageway 11, and an operating grip (ribs) 12 which areintegrally formed.

The piercing pipe 10 is used for charging the reactor tube 1 with asample and a reagent solution and feeding pressurized air for applying apressure to the interior of the reactor tube 1. Preferably this piercingpipe 10 is tapered, i.e. progressively reduced in inner diameter, towardthe reactor tube 1. The valve passageway 11 has one end connectableliquid-tight to the discharging pipe 5 in the axial direction of thereactor tube 1 and the other end adapted for liquid-tight connection toa flow passageway communicating with the interior of the drain cup 2,thus forming a drain passageway for discharging the reaction mixturefrom the reactor tube 1 to the drain cup 2. The rotary element 6 is soconstructed that as the grip 12 is rotated, the element 6 is rotatedabout the axis of the reactor tube 1. Assuming, by way of illustration,that the rotary element 6 is in the position shown in FIG. 2, thereaction mixture solution in the reactor tube 1 can be allowed to draininto the drain cup 2 through the drain passageway extending from thereactor tube 1 through the discharging pipe 5 and valve passageway 11 tothe drain cup 2. As the rotary element 6 is rotated through 180 degreesabout the axis of the reactor tube 1 from the above position, the flowpassageway consisting of the discharging pipe 5 and valve passageway 11is brought into communication with the recovery tube 3 through a flowpassageway formed by the channel cover 8, as illustrated in FIG. 4, sothat the reaction product in the reactor tube 1 can be withdrawn intothe recovery tube 3.

In this manner, the channel cover 8 not only covers the top opening ofthe recovery tube 3 but also provides at least a part of the flowpassageway for discharging the objective nucleic acid from the reactortube 1 into the recovery tube 3.

The cover 7 disposed across the top opening of the drain cup 2 ispreferably provided with a hole therein so that an exhaust nozzle may beconnected thereto when the reaction mixture is to be discharged from thereactor tube 1 into the drain cup 2.

The extraction vessel may be molded from virtually any material and suchconventional materials as glass, polypropylene, polyvinyl chloride, andsilicone-coated general resin, among others, can be employed. Amongthose materials, polypropylene is particularly preferred because theobjective nucleic acid will not be adsorbed thereon.

The geometry of the extraction vessel is not particularly restricted.For example, the reactor tube 1 may be a conical one, the drain cup 2may be cylindrical, and the recovery tube 3 may be shaped like anordinary microtube, for example a tube having a conical bottom.

2. Nucleic acid extraction apparatus

The construction of the nucleic acid extraction apparatus of theinvention is now described.

FIG. 5a and FIG. 5b are a top view and a side view, respectively, of anucleic acid extraction apparatus embodying the principles of theinvention. In those views, the reference numeral 13 represents a vesselstand on which said group of extraction vessels is mounted, 14 adistribution means having a distribution nozzle for dispensing thesample and reagent solutions into the reactor tube of said extractionvessel and a rotary arm for rotating said rotary element 6. Thereference numeral 15 represents a stirring means for admixing thesolutions in the reactor tube 1 of said extraction vessel, 16 representsa discharging means for withdrawing the solution in said reactor tube 1into said drain cup 2 or said recovery tube 3, and 17 represents anintegral unit comprising a holding means for holding a magnetic carrierstationary on the internal wall (internal bottom area) of the reactortube 1 and a heating means for heating the solution in the reactor tube1, which works in contacting with the external wall (external bottomarea) of the reactor tube 1.

The reference numeral 18 represents a reagent bottle or bottle groupcontaining the reagents (extraction solvent, wash solution, eluent,etc.) for use in the nucleic acid extraction procedure.

The reference numerals 19, 20, 21 and 22 taken together represent ameans for transfer of the vessel stand 13. Thus, the numeral 19represents a motor, 20 a continuous belt, 21 a rail, and 22 a pulley.Thus, the vessel stand 13 carrying the extraction vessel traverses alongthe rails 21 as the motor 19 and, hence, the belt 20, and pulley 22 aredriven, and as shown in FIG. 5b, successively occupies the positions A,B, C, and D where said distribution means 14, stirring means 15, holdingmeans 17 a and discharging means 16, and heating means 17 b arerespectively disposed. In this specification, those means arecollectively referred to as a transfer means.

The apparatus of the invention may further include a means fortransferring extraction vessels to the vessel stand and disposing themthereon.

The detailed structure of the means represented by the referencenumerals 14-17 are now described.

FIG. 6 is a schematic diagram illustrating the distribution means 14 fordispensing solutions into the reactor tube 1 of the extraction vessel.The mechanical components of this distribution means 14 are mounted on aliner motor base 26 so that they may traverse along rails 25horizontally. Connected to the motor 23 is a pole screw 29, whereby amovable piece 31 is caused to move up and down in response to rotationof the pole screw. This movable piece 31 is symmetrically flanked by apair of L-shaped metals 33 and the distribution nozzle 34 is attached toone of them, while the rotary arm 27 is attached to the other L-shapedmetal.

Fitted to the forward end of the distribution nozzle 34 is a resin pad35 shaped to fit the bore of the piercing pipe 10 of the rotary element6 so that said forward end may come into connection with the inlet tothe reactor tube 1, that is to say the piercing pipe 10 of the rotaryelement 6. The reference numeral 32 represents a spring and thereference numeral 30 represents a tube for feeding the reagent and othersolutions from the reagent bottle or bottle group 18, said tube 30 beingconnected to the distribution nozzle 34 in a liquid-tight manner. Therotary arm 27 attached to the L-shaped metal 33 on the opposite side tothe distribution nozzle 34 is adapted to rotate the rotary element 6.This arm 27 has a rotary arm grip 28 shaped to fit the grip 12 of therotary element 6 at its forward end and is driven by a motor 24.

FIG. 7 is a schematic view showing the stirring means 15 for stirringthe solution within the reactor tube 1 of the extraction vessel. Thestirring is effected as a ring-shaped permanent magnet (ring magnet) 36reciprocates vertically with the reactor tube 1 being included in itscentral hole. The reference numeral 37 represents a motor, 40 a polescrew, and 38 and 39 each represents a fixing plate supporting thestirring mechanism. As the fixing plate 39 reciprocates vertically inresponse to rotation of the pole screw 40, the ring magnet 36 secured tothis fixing plate is also caused to reciprocate vertically insynchronism.

FIG. 8 is a schematic diagram showing an integral unit 17 comprising aholding means and a heating means. The holding means 17 a is comprisedof a permanent magnet 41 by which the magnetic carrier contained in thereactor tube 1 of the extraction vessel is retained on the inside wall(inside bottom wall) of the reactor tube. The heating means 17 bcomprises a heating block 42 made of a metal such as aluminum and havinga recess complementary to a bottom portion of the reactor tube 1 foraccepting said bottom portion and a platform 44 having a built-in planeheater (not shown) for controlling the temperature of said heatingblock. The integral unit 17 includes a motor 45, a pole screw 48, andfixing metals 43, 46 and 47 for vertically reciprocating the permanentmagnet 41, heating block 42 and platform 44.

FIG. 9 is a schematic view showing the discharging means 16 positionedon the extraction vessel. Since this discharging means 16 must also bemovable vertically, it comprises a motor 49, a pole screw 50 and amovable piece 51 adapted to move up and down in response to rotation ofsaid pole screw. This discharging means further comprises a pressurenozzle 53 and an exhaust nozzle 57 as attached to an L-shaped metal 52connected to said movable piece 51. The forward end of the pressurenozzle 53 carries a resin pad 54 complementary to the inside geometry ofthe inlet to the reactor tube 1 of the extraction vessel, that is to saythe piercing pipe 10 of the rotary element 6. This pressure nozzle 53 isconnected air-tight to a pressurized air supply tube 55 through a jointmetal 56.

On the other hand, the forward end of the exhaust nozzle 57 is fittedwith a resin pad 58 conforming to a hole in the cover 7 mounted on thetop opening of the drain cup 2. This exhaust nozzle 57 is connectedair-tight to a tube 59 for discharging air from the drain cup 2 to adisposal bottle (not shown) through a joint metal 60.

The nucleic acid extraction apparatus of this invention basicallycomprises said various means (distribution means, stirring means,holding means, discharging means, heating means, and transfer means) andthose means are controlled by a computer (not shown).

3. Nucleic acid extraction method

The method of extracting a nucleic acid using the above-describednucleic acid extraction apparatus of the invention is now described. Thenucleic acid extraction method using the above apparatus of theinvention can be carried out by the technique utilizing a magneticcarrier as described in Unexamined Japanese Patent Publication No.19292/1997, for instance. The published literature is incorporatedherein by reference.

This nucleic acid extraction method comprises feeding magnetic silicabeads containing a superparamagnetic metal oxide, a sample containingthe objective nucleic acid, and a solvent for nucleic acid extractioninto the extraction vessel, mixing them together to let the nucleic acidattached to the magnetic silica beads, applying a magnetic field to thevessel to separate the magnetic silica beads carrying the nucleic acidfrom the solution (B/F separation), and finally eluting the nucleic acidfrom the magnetic silica beads.

The sequence of steps according to this nucleic acid extraction method(flow-chart) is shown in FIG. 10. The method of using the nucleic acidextraction apparatus of the invention is now described in accordancewith this flow-chart. Schematic diagrams pertinent to the respectivesteps are shown in FIGS. 11-16.

First, the reactor tube 1 of the extraction vessel is charged with thesample (for example a biological sample such as blood) and the magneticsilica beads for attraction of nucleic acid. In consideration of safety,this procedure is generally carried out by the operator in a clean benchor the like. During this procedure, the reagent solution for extractionis fed to the reactor tube.

The extraction vessel thus containing the sample, magnetic silica beads,and extraction reagent solution in the reactor tube is transferred andinstalled on the vessel stand 13 of the nucleic acid extractionapparatus (FIGS. 5a and 5 b). This transfer and installation of theextraction vessel on the vessel stand 13 can be carried out utilizingthe vessel transfer and installation means with which the extractionapparatus is optionally provided.

The foregoing represents a preparation stage preceding the operation ofthe apparatus of this invention. After the above installation of theextraction vessel, the nucleic acid extraction procedure is started, forexample by pressing a start button (not shown). The first step in thisprocedure is a stirring step where the solution in the reactor tube 1 isstirred to extract nucleic acid from the sample and cause it to beadsorbed on the magnetic silica beads as described in UnexaminedJapanese Patent Publication No. 19292/1997.

The vessel stand 13 is transferred, by said transfer means, to positionB where the stirring means 15 is located (FIG. 5b). The vessel standremains stationary in this position during the solution in the reactortube 1 is stirred for thorough mixing. As shown in FIG. 12, thisstirring and mixing is effected as the ring magnet 36 of the stirringmeans 15 reciprocates vertically with the reactor tube 1 beingpositioned within its central hole to thereby cause the magnetic silicabeads in the reactor tube 1 to move up and down. The stroke ofreciprocation of the ring magnet 36 and the stirring time can bejudiciously selected as necessary.

On completion of stirring, the vessel stand 13 is transferred, again bysaid transfer means, to position C where the holding means 17 is located(FIG. 5b). The vessel stand remains stationary in this position and thenext step, i.e. B/F separation, is carried out. This status is shown inFIG. 13. The permanent magnet 41 of the holding means contacts theexterior bottom of the reactor tube 1 of the extraction vessel, wherebythe magnetic silica beads 61 in the reactor tube 1 are retained in thebottom area of the reactor tube.

Furthermore, in this position C, the discharging means 16 is set abovethe extraction vessel and the next step, i.e. discharge of the unwantedcomponent solution, is carried out. Thus, with the B/F separation beingperformed as shown in FIG. 13, the pressure nozzle 53 and exhaust nozzle57 constituting the discharging means descend into engagement with thepiercing pipe 10 and the hole of the cover 7, respectively, of theextraction vessel. This status is shown in FIG. 14.

As shown in FIG. 9, the pad 54 fitted to the forward end of the pressurenozzle 53 is inserted air-tight into the inlet of the reactor tube 1,that is to say the piercing pipe 10, and the pressurized air from a pump(not shown) is blown into the reactor tube 1. As a result, the unwantedcomponent solution in the reactor tube 1 is discharged into the draincup 2 through the discharge passageway comprised of the discharging pipe5, valve passageway 11, etc. At the same time, the air introduced underpressure into the drain cup 2 is exhausted through the hole in the cover7 on the drain cup and the passageway constituted by the exhaust nozzle57. This series of B/F separation and discharge of the unwantedcomponent solution is performed in position C.

Upon completion of discharge of the unwanted component solution, thedischarging means 16 and holding means 17 a return to a given position.On the other hand, the vessel stand 13 moves again to position A forintroduction of the next solution (wash solution) into the reactor tube1 and stops there (FIG. 5b). In accordance with the flow-chart shown inFIG. 10, the washing step is now carried out.

Introduction of the wash solution is performed by the distribution means14 which serially dispenses the wash solution into the reactor tubes 1of extraction vessels arranged on the vessel stand 13. As shown in FIG.6, the pad 35 fitted to the forward end of the distribution nozzle 34 isinserted liquid-tight into the inlet of the reactor tube (the piercingpipe 10) and the reagent solution (wash solution) is introduced from thebottle 18 into the reactor tube 1 through the tube 30. Upon completionof dispensing into one extraction vessel, the distribution means 14moves to the next extraction vessel and dispenses the wash solution inthe same manner. Upon completion of dispensing into all the extractionvessels arranged on the vessel stand 13, the distribution means 14returns to a given position and stops there.

The subsequent operations in the washing procedure, i.e. stirring, B/Fseparation, and discharge of the unwanted solution, may be performed inexactly the same manner as in the above extraction procedure. Thewashing procedure in nucleic acid extraction may vary, for exampleinvolving change of wash reagents or a plurality of washing cycles usingthe same reagent. In any case, the washing operations by the apparatuscan be exactly the same as those described above.

The drying of the magnetic silica beads is now described.

Upon completion of discharge of the unwanted solution (washings) formedin the washing step, the vessel stand 13 is shifted to position D (FIG.5b). Disposed in position D is the heating block 42 constituting theheating means 17 b. As the heating block 42 ascends in response torotation of the pole screw 48, the reactor tube 1 of the extractionvessel comes into contact with its recessed part and is heated. Thedrying is effected as the reactor tube is heated until the residual washsolution on the surface of the magnetic silica beads in the reactor tubehave been completely evaporated off (FIG. 15).

Upon completion of drying, the distribution means 14 dispenses asolution of low ion concentration (eluent), such as sterilized water,into the reactor tube containing the magnetic silica beads and thesolution is warmed for a definite time. This warming of the eluent isalso performed by the heating block 42 disposed in position D (FIG. 5b).FIG. 15 shows this drying or eluent warming step.

Upon completion of warming of the eluent, the heating block 42 returnsto a given position and the vessel stand 13 is shifted to position Bwhere the stirring means 15 is disposed and the stirring is carried out.

Upon completion of stirring, the final nucleic acid recovery step isexecuted. For this purpose, the vessel stand 13 is shifted to position Awhere the distribution means 14 is located (FIG. 5b). Then, by therotary arm 27 of the distribution means 14, the rotary element 6 mountedon the reactor tube 1 is rotated through 180 degrees. Thereupon, asillustrated in FIG. 4, the flow passageway constituted by the channelcover 8 of the recovery tube is brought into communication with thevalve passageway 11 and discharging pipe 5 in the reactor tube 1 so thata preparation for recovery of the nucleic acid is completed. The aboverotation of the rotary element is effected as the rotary arm grip 28 isengaged with the grip 12 of the rotary element 6 and the rotary arm 27is driven by the motor 24. This rotation is serially carried out for oneextraction vessel after another.

Upon completion of rotation of the rotary element 6 for all theextraction vessels, the vessel stand 13 is shifted to position C wherethe holding means 17 a is disposed (FIG. 5b). Here, as shown in FIG. 16,the magnetic silica beads are held stationary by the permanent magnet 41for B/F separation and the pressurized air is introduced via thepressure nozzle 53 into the reactor tube 1. By this pressurization, theeluate containing the nucleic acid is discharged into the recovery tube3 through the discharging pipe 5 and valve passageway 11 to recover thenucleic acid.

Upon completion of this recovery, the vessel stand 13 is shifted fromposition C to a given initial position, whereby the entire nucleic acidextraction procedure is completed and the apparatus stops its actions.Then, the operator takes out the extraction vessels from the vesselstand 13, disconnect the recovery tubes 3, and preserves them in therefrigerator or otherwise takes the necessary procedure.

The nucleic acid extraction procedure using the nucleic acid extractionapparatus of the invention has by now been described.

As mentioned hereinbefore, the extraction vessel and nucleic acidextraction apparatus of this invention are the vessel and apparatuswell-suitable with the nucleic acid extraction technology utilizing anucleic acid-binding magnetic carrier and, therefore, are simple andcompact as compared with the system utilizing a centrifuge or a vacuumpump.

Furthermore, since in the apparatus of the invention the respectivesteps in the nucleic acid extraction procedure (dispensing of solutions,stirring, discharging and heating steps) are carried out at the definitepositions for the means (distribution means, stirring means, dischargingmeans and heating means) with one-to-one correspondence, by transferringextraction vessels serially thereto, the complicated extractionprocedure can be carried through with high reliability, without risksfor contamination, rapidly, and with high efficiency.

Moreover, since the extraction vessel of this invention is not open tothe external atmosphere, the risk for contamination due tovessel-to-vessel carryovers is avoided so that highly dependable resultscan be expected. In addition, when the extraction vessels are disposablevessels, the cleaning operation can be omitted to avoid the risk forcontamination by wash residues and the risk for infection with theviruses which biological samples may contain.

The apparatus of this invention thus features high dependability andsafety.

Therefore, in the field of clinical examinations for genetic diagnosiswhere a large number of samples must be dealt with at one time as wellas in the field of basic research, the present invention provides anucleic acid extraction apparatus of great utility value with whichnucleic acid components can be extracted rapidly, expediently, safely,and with high dependability.

What is claimed is:
 1. A nucleic acid extraction apparatus comprising:(1) a group of extraction vessels each comprising a reactor tube inwhich a sample, a reagent solution and a magnetic carrier are admixedand reacted, a drain cup for pooling an unwanted component solution anda nucleic acid recovery tube, all as secured to a supporting plate, (2)a distribution means for dispensing a sample or other solution into eachof said extraction vessels, (3) a stirring means for stirring the samplesolution and magnetic carrier within the extraction vessel, (4) aholding means for holding said magnetic carrier stationary in a positionwithin the extraction vessel, (5) a discharging means for dischargingthe sample solution from the reactor tube into the drain cup or nucleicacid recovery tube while said magnetic carrier is held stationary withinthe reactor tube, (6) a heating means for heating the sample solutioncontaining the magnetic carrier in said extraction vessel, and (7) atransfer means for transferring said extraction vessel to the requiredpositions where said distribution means, stirring means, holding means,discharging means, and heating means are respectively disposed.
 2. Anucleic acid extraction apparatus comprising: (1) a vessel stand adaptedto support a group of extraction vessels each comprising a reactor tubeto be filled with a magnetic carrier, a sample and a reagent solution, adrain cup and a nucleic acid recovery tube, all as secured to asupporting plate; (2) a distribution means for dispensing a sample orother solution into the reactor tube of said extraction vessel installedon said vessel stand; (3) a stirring means for stirring the magneticcarrier, sample, and reagent solution within the reactor tube; (4) aholding means for holding the magnetic carrier stationary within thereactor tube; (5) a discharging means for discharging the solution inthe reactor tube into the drain cup or nucleic acid recovery tube whilethe magnetic carrier is held stationary within the reactor tube; (6) aheating means for heating the reactor tube containing at least saidmagnetic carrier; (7) a transfer means for transferring the vessel standcarrying the extraction vessels horizontally; and (8) a control meansfor controlling said respective means.
 3. A nucleic acid extractionapparatus according to claim 2 wherein the control means has a functionto control the transfer means in such a manner the vessel stand may betransferred to any of the positions where said distribution means,stirring means, holding means, discharging means, and heating means arerespectively disposed.
 4. A nucleic acid extraction apparatus accordingto claim 2 further comprising a means for transferring the extractionvessels and installing them in an array on the vessel stand.
 5. Anucleic acid extraction apparatus according to claim 2 wherein theextraction vessel for installing in the vessel stand comprising: areactor tube, a drain cup, and a nucleic acid recovery tube as securedto a supporting plate, a channel cover adapted to provide a connectingpath between the nucleic acid recovery tube and the reactor tube asmounted in a top opening of said recovery tube, a cover with a hole forconnection of an exhaust nozzle as mounted air-tight in a top opening ofsaid drain cup, a rotary element comprising a piercing pipe forinsertion and connection of a distribution nozzle, a valve passagewayadapted to provide a connecting path between the reactor tube and thedrain cup or the recovery tube, and a rotating grip as mounted air-tightand axial-rotably in a top opening of said reactor tube, and one end ofsaid valve passageway being connected liquid-tight to a discharging pipeextending axially within the reactor tube and the other end beingconnected air-tight to a passageway communicating with the interior ofthe drain cup.
 6. A nucleic acid extraction apparatus according to claim5 wherein at least the nucleic acid recovery tube of the extractionvessel is detachable from the reactor tube and drain cup.
 7. A vesselfor nucleic acid extraction which comprises: a reactor tube, a draincup, and a nucleic acid recovery tube as secured to a supporting plate,a channel cover adapted to provide a connecting path between the nucleicacid recovery tube and the reactor tube as mounted in a top opening ofsaid recovery tube, a cover with a hole for connection of an exhaustnozzle as mounted air-tight in a top opening of said drain cup, a rotaryelement comprising a piercing pipe for insertion and connection of adistribution nozzle, a valve passageway adapted to provide a connectingpath between the reactor tube and the drain cup or the recovery tube,and a rotating grip as mounted air-tight and axial-rotably in a topopening of said reactor tube, and one end of said valve passageway beingconnected liquid-tight to a discharging pipe extending axially withinthe reactor tube and the other end being connected air-tight to apassageway communicating with the interior of the drain cup.
 8. Anucleic acid extraction vessel according to claim 7 wherein at leastsaid nucleic acid recovery tube is detachable from said reactor tube anddrain cup.